Considered to be the largest organ of the body, the primary function of skin is to act as a barrier between the organism and the environment. Strong cell-cell junctions, formed by adherens junctions, tight junctions, and desmosomes, are critical to the integrity of the epidermis and its ability to resist mechanical stress. Desmosome-targeting genetic, autoimmune, and infectious diseases present clinically in both the skin and heart, two organs with tissues subjected to significant mechanical forces, which suggest that a major function of desmosomes is to resist mechanical stress. While it has been shown that expression of desmosomal and keratin proteins are critical to the mechanical integrity of skin, it is not known if desmosomes act primarily as mechanical or signaling molecules. Because the mechanical force applied to desmosomes has never been directly measured it is also not known if therapies that strengthen desmosomes would be a successful strategy for treatment of skin-blistering and wound healing. The central hypotheses of this proposal is that desmosomes are subject to tensile forces applied by the keratin cytoskeleton, and that the level of tension is altered in wound healing and skin diseases. The major innovation in this project is the use of a new technique developed by my lab to directly measure desmosome tension through the use of desmoglein-2 and -3 FRET- based tension sensors. This novel approach will provide significant insight into the magnitude and regulation of desmosome forces, the ability of the IF cytoskeleton to transmit and apply mechanical force, and the role of desmosome forces in skin physiology and pathology. In Aim 1, the dynamics of desmosome tension will be examined during the process of desmosome formation, and regulators of desmosome tension will be identified. Additionally, changes in desmosome tension will be measured in cells subjected to cyclic stretch. In Aim 2, desmosome tension will be measured during the process of wound healing and also in epithelial to mesenchymal transition. In Aim 3, in vitro models of pemphigus vulgaris and epidermolysis bullosa simplex will be used to determine if desmosome tension is altered in skin blistering diseases. We will address a significant gap in the understanding the role of tissue strength in diseases with skin blistering and wounding diseases caused by mutations in or autoimmunity to desmosomes or keratin IFs. Namely, we will address if the principal role of desmosomes is mechanical or signaling. These basic mechanobiology studies will provide significant insight into the dynamics of mechanical forces across desmosomes under conditions of normal homeostasis and disease, and are essential to the identification of new therapeutic targets for wound healing and desmosome-related skin pathologies. Additionally, the role of desmosomes in force transmission may also provide mechanistic insight into why desmosome-associated diseases also frequently present as cardiomyopathies. Lastly, our understanding of the ability of IF to transmit mechanical forces is lacking. As a result, these studies will fundamentally impact the understanding of cellular biomechanics.